Diagnosing and treating hormone resistant cancers

ABSTRACT

Provided herein are methods and compositions related to diagnosing and treating hormone resistant cancers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/912,752, filed Apr. 19, 2007, which is incorporated by referenceherein in its entirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was funded by the National Institutes of Health (GrantNos. 5T32CA086800-04 and CA098301-01) and the U.S. Army Department ofDefense (Grant No. DAMD17-02-1-0584). The government has certain rightsin this invention.

BACKGROUND

The response of receptors to hormones is particularly important in thedevelopment of a number of diseases, including cancer. Hormone resistantcancers include certain breast, endometrial, ovarian and prostatecancers.

Breast cancer is the leading cause of death among American women betweenthe ages of 20 and 59. Among a variety of established etiologicalfactors linked to breast cancer, the steroid hormone estrogen(17-β-estradiol; E2) has long been implicated in disease pathogenesis.Numerous animal studies have revealed that E2 can induce and promotebreast cancer, while estrogen ablation therapy or the administration ofantiestrogens can oppose these effects. The physiological effects of E2in the breast are mediated by cognate receptors that are expressed astwo structurally related subtypes, estrogen receptor α (ERα) and β(ERβ). ERα is the predominant receptor isoform expressed in breastcancer cells, and approximately 70% of breast cancer patients scorepositive for ERα at diagnosis. ERα is therefore a predictive factor withrespect to breast cancer development and hormone sensitivity status.Endocrine therapy, which seeks to block ER-mediated mitogenic signaling,has emerged as one of the most important systemic therapies in breastcancer management. However, therapeutic resistance, either inherent (denovo resistance) or acquired during treatment (acquired resistance)remains a significant clinical roadblock to effective diseasemanagement.

Prostate cancer is the second leading cause of cancer death among malesin the United States. Although survival rates are good for prostatecancer that is diagnosed early, the treatments for advanced disease arelimited to hormone ablation techniques and palliative care. Hormoneablation techniques (orchiectomy and anti-androgen treatments) generallyallow only temporary remission of the disease. It usually recurs within1-3 years of treatment, with the recurrent tumors no longer requiringandrogens for growth and survival. Therapy with conventionalchemotherapeutic agents, such as progesterone, estramustine andvinblastine, has also not been demonstrated to be effective to haltprogression of the disease.

SUMMARY

Provided herein are methods and compositions related to diagnosing andtreating hormone resistant cancers. Specifically, provided is a methodof determining whether a cancer cell is sensitive to endocrine therapyby determining the level of expression or activity of Deleted in BreastCancer-1 (DBC-1) or the presence of binding of DBC-1 and EstrogenReceptor α (ERα) in the cell. Also provided is a method of determiningwhether a subject with cancer is suitable for treatment with endocrinetherapy comprising determining the level of expression or activity ofDBC-1 or the presence of binding of DBC-1 and ERα.

Provided is a method of determining a susceptibility to hormoneresistant cancer in a subject comprising determining the level ofexpression or activity of DBC-1 or the presence of binding of DBC-1 andERα. Also provided is a method of inducing apoptosis of cancer cellscomprising selecting a population of hormone resistant cancer cells andcontacting the hormone resistant cancer cells with an agent thatinhibits expression of DBC-1 or binding of DBC-1/ERα.

Provided is a method of treating hormone resistant cancer in a subject,comprising selecting a subject with hormone resistant cancer andadministering an agent that inhibits expression of DBC-1 or binding ofDBC-1/ERα to the subject.

The details of one or more embodiments are set forth in the accompanyingdrawings and the description below. Other features, objects, andadvantages will be apparent from the description and drawings, and fromthe claims.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-D show DBC-1 and ERα interact in vivo in a ligand-independentmanner. FIG. 1A shows mammalian two-hybrid interaction analysis. HeLacells cultured in hormone-free medium for three days were transfectedwith the indicated combinations of mammalian expression plasmidsencoding the yeast GAL4 DNA-binding domain (GAL4), the Herpes simplexvirus VP16 transactivation domain (VP16), a GAL4-DBC-1 chimera, and aVP16-ERα chimera. Twenty-four hours post-transfection cells were treatedwithout (−E2) or with (+E2) 17-β-estradiol (10⁻⁷ M) for an additionaltwenty-four hours prior to cell harvest and assay of transfected wholecell lysates for luciferase activity produced from a co-transfected GAL4DNA-binding site driven-reporter template. Luciferase values areexpressed relative to the luciferase activity obtained in cellstransfected with both the GAL4 and VP16 expression vectors, which wasarbitrarily assigned a value of 1. Luciferase activities were firstnormalized to (3-galactosidase activity obtained by cotransfection of aβ-galactosidase expression vector. Error bars represent the standarddeviation (S.D.) from the average of at least three independenttransfections performed in duplicate. Note that estrogen abolishes theinteraction between GAL4-DBC-1 and VP16-ERα. FIG. 1B shows mammaliantwo-hybrid interaction analysis. For FIG. 1B, top panel, HeLa cellscultured for three days in hormone-free medium (−E2) were transfectedwith the indicated combinations of mammalian expression plasmidsencoding GAL4, VP16, a GAL4-DBC-1-N terminal chimera (amino acids1-478), a GAL4-DBC-1-C terminal chimera (amino acids 479-923), and aVP16-ERα chimera. Forty-eight hours post-transfection, cells wereharvested and transfected whole cell lysates were assayed for luciferaseactivity produced from a cotransfected GAL4 DNA-binding sitedriven-reporter template as described in FIG. 1A. Note that ERαinteracts exclusively with the N-terminus of DBC-1. For FIG. 1B, bottompanel, harvested whole cell lysates were resolved by SDS-12%-PAGE andprocessed by immunoblot analysis with antibodies specific for GAL4-DBDor ERα as indicated by arrows. Note that differences in the relativeexpression levels of the GAL4-DBC-1 chimerae cannot explain differencesin their respective ERα-binding capabilities. Results are representativeof at least three independent experiments.

FIGS. 1C and 1D show co-immunoprecipitation analysis. For FIG. 1C MCF-7cells cultured in hormone-free medium for three days were treatedwithout (−E2) or with (+E2) 17-β-estradiol (10⁻⁷ M) for one hour priorto cell harvest and immunoprecipitation of whole cell lysates withantibodies specific for ERα (top panel) or DBC-1 (bottom panel).Immunoprecipitates were resolved by SDS-10%-PAGE and processed byimmunoblot analysis using antibodies specific for DBC-1 or ERα asindicated by arrows. Note specific immunoprecipitation of DBC-1 byERα-specific antibodies and ERα by DBC-1-specific antibodies only in theabsence, but not in the presence, of estrogen. Results arerepresentative of at least three independent experiments. For FIG. 1D,T-47D (top panel) and BG-1 (bottom panel) cells cultured in hormone-freemedium for three days were treated without (−E2) or with (+E2)17-β-estradiol (10⁻⁷ M) for one hour prior to cell harvest andimmunoprecipitation of whole cell lysates with antibodies specific forERα. Immunoprecipitates were resolved by SDS-10%-PAGE and processed byimmunoblot analysis using antibodies specific for DBC-1 or ERα asindicated by arrows. Results are representative of at least threeindependent experiments.

FIGS. 2A and 2B show via coimmunoprecipitation analysis that DBC-1 andunliganded ERα associate in the nucleus independently of HSP90. For FIG.2A, MCF-7 cells cultured in hormone-free medium for three days weretreated without (−E2) or with (+E2) 17-β-estradiol (10⁻⁷ M) for one hourprior to cell harvest and immunoprecipitation of whole cell lysates withantibodies specific for DBC-1. Immunoprecipitates were resolved bySDS-10%-PAGE and processed for immunoblot analysis with antibodiesspecific for DBC-1, HSP90, ERα, or CYP40 as indicated by arrows. Resultsare representative of at least three independent experiments. For FIG.2B, MCF-7 cells cultured in hormone-free medium for three days werefractionated into cytoplasmic (cyto) and nuclear (nuc) extracts.Equivalent amounts of each extract were immunoprecipitated withantibodies specific for ERα. Immunoprecipitates were resolved bySDS-10%-PAGE and processed for immunoblot analysis with antibodiesspecific for DBC-1 or ERα as indicated by arrows. Note that anadditional immunoprecipitation containing four times the amount ofcytoplasmic extract (4× cyto) failed to yield a detectable amount ofDBC-1 in either the input or immunoprecipitate. Results arerepresentative of at least three independent experiments.

FIGS. 3A and 3B show via GST-pulldown assays that the DBC-1 N-terminusbinds to the ERα hormone-binding domain in vitro. GST pulldown assayswere performed using full-length in vitro translated DBC-1 and GST-ERαfragments (FIG. 3A) or in vitro translated DBC-1 fragments andGST-full-length ERα (FIG. 3B) as indicated. Numbers refer to amino acidcoordinates. 35S-labeled in vitro translated proteins were incubatedwith glutathione-sepharose immobilized GST derivatives and boundproteins were resolved by SDS-12%-PAGE prior to detection byPhosphorimager analysis. Input represents 10% of the 35S-labeled invitro translated proteins used in binding reactions. The amount of eachDBC-1 derivative retained by GST-ERα (% bound) was quantified andexpressed as a percentage of the total input. Percent (%) bound refersto the average and S.D. of at least three independent experiments.Asterisks denote statistically significant (p<0.05) binding valuesrelative to GST alone. Note that DBC-1 binds primarily to GST-ERαderivatives 1-595 (full-length ERα) and 302-595 (ERα hormone-bindingdomain), while GST-ERα binds primarily to DBC-1 derivative 1-150(N-terminus). Schematic diagrams of ERα and DBC-1 indicate fragmentsused in binding reactions. Abbreviations used are AF-1, activationfunction 1; DBD, DNA-binding domain; AF-2/HBD, activation function2/hormone-binding domain; NLS, putative nuclear localization sequence;LZip, putative leucine zipper.

FIGS. 4A and 4B show that tamoxifen and ICI 182,780 disrupt theinteraction between DBC-1 and ERα. MCF-7 (FIG. 4A) or BG-1 (FIG. 4B)cells cultured in hormone-free medium for three days were treated withvehicle (−E2), 17-β-estradiol (10⁻⁷ M; +E2), 4-hydroxytamoxifen (10⁻⁶ M;4-OHT), or ICI 182,780 (10⁻⁷ M; ICI) for one hour prior to cell harvestand immunoprecipitation of whole cell lysates with antibodies specificfor ERα. Immunoprecipitates were resolved by SDS-7.5%-PAGE and processedfor immunoblot analysis with antibodies specific for DBC-1 or ERα asindicated by arrows. Results are representative of at least threeindependent experiments.

FIGS. 5A and 5B show that RNAi-mediated DBC-1 suppression is accompaniedby reduced steady-state levels of unliganded ERα. MCF-7 cells culturedin hormone-free medium for three days were electroporated with control(siCNTL) or DBC-1-specific (siDBC-1) siRNA (21 nM) as indicated.Electroporated cells were cultured without (−E2) or with (+E2)17-β-estradiol (10⁻⁷ M) for an additional three days prior to cellharvest. For FIG. 5A, harvested whole cell lysates were resolved bySDS-10%-PAGE and processed by immunoblot analysis with antibodiesspecific for DBC-1, ERα, or TFIIEβ as indicated by arrows. Results arerepresentative of at least three independent experiments. For FIG. 5B,top panel, immune signals were quantified using a Kodak ImageStation2000R. ERα protein levels were normalized to TFIIEβ and plotted relativeto the ERα protein level in control siRNA cells cultured in the absenceof E2, which was arbitrarily assigned a value of 1. Error bars representthe S.D. from the average of at least three independent experiments. ForFIG. 5B, bottom panel, RNA was processed by quantitative RT-PCR analysisfor the levels of DBC-1, ERα, and GAPDH mRNAs. ERα RNA levels werenormalized to GAPDH levels, and expressed relative to the level ofERαRNA in control siRNA cells cultured in the absence of E2, which wasarbitrarily assigned a value of 1. Error bars represent the S.D. fromthe average of at least three independent experiments performed induplicate.

FIG. 6 shows RNAi-mediated DBC-1 depletion inhibits estrogen-independentproliferation in human breast cancer cells. MCF-7 cells cultured inhormone-free medium for three days were electroporated with control orDBC-1-specific siRNA (21 nM) as indicated and cultured without (−E2) orwith (+E2) 17-β-estradiol (10⁻⁷ M). Culture medium was replaced everytwo days. Cell proliferation was monitored by counting with trypan blueexclusion for seven days following electroporation. p-values are incomparison to controls. Error bars represent the S.D. from the averageof at least three independent experiments performed in triplicate.

FIGS. 7A and 7B show that DBC-1 is an ERα-dependent prosurvival factorin human breast cancer cells. MCF-7 (FIG. 7A) or MDA-MB-231 (FIG. 7B)cells cultured in hormone-free medium for three days were electroporatedwith control or DBC-1-specific siRNA (21 nM) as indicated. Forty-eighthours following electroporation, cells were treated with vehicle (−E2),17-β-estradiol (10⁻⁷ M; +E2), ICI 182,780 (10⁻⁷ M; ICI), or acombination of E2 and ICI 182,780 (E2+ICI) for an additional twenty-fourhours prior to cell harvest. For FIGS. 7A and 7B, Top panels, harvestedcells were stained with Annexin V-FITC and propidium iodide prior toquantification of apoptosis by flow cytometric analyses. p-values are incomparison to controls. Error bars represent the S.D. from the averageof at least three independent experiments performed in triplicate. ForFIGS. 7A and 7B, Bottom panels, cell lysates from representativeapoptosis assays in panels (FIG. 7A) and (FIG. 7B) were resolved bySDS-10%-PAGE and processed by immunoblot analysis with the indicatedantibodies specific for DBC-1 or TFIIEβ as a loading control.

FIGS. 8A, 8B and 8C show biochemical purification of ligand-independentERα-associated proteins (ERAPs). In FIG. 8A, nuclear extracts (NEXT)from hormone-deprived fERα/S3 cells treated without (−E2) or with (+E2)17-β-estradiol (10⁻⁷ M) for twenty-four hours were chromatographed inparallel on phosphocellulose (PC-11) columns using a step gradientelution of increasing salt (KCl) concentration. Individual stepfractions (40 pg) were resolved by SDS-10%-PAGE and processed byimmunoblot analysis using an ERα-specific antibody (HC-20). FIG. 8Bshows a fractionation and immunopurification scheme fromhormone-deprived fERalS3 and parental HeLaS3 cell nuclear extracts. InFIG. 8C, 0.3 M KCl step fractions derived from PC-11 chromatography ofhormone-deprived fERα/S3 and HeLaS3 cell nuclear extracts (input) weresubjected, in parallel, to anti-FLAG M2 monoclonal antibody affinitychromatography and elution with FLAG peptide (FLAG eluate). Peptideeluates were resolved by SDS-10%-PAGE and processed by silver staining.Additionally, size-selected ERAP pools were subjected to massspectrometric based-peptide sequence analysis. Arrows identify ERα andDBC-1.

FIGS. 9A-9L are pictures of representative immunohistochemical stainingof DBC-1 in normal and cancerous breast tissues. (A-D) DBC-1 staining innormal breast tissue. (E-H) Enlarged images of (A-D), respectively.(I-L) DBC-1 staining in invasive breast cancer. (M-P) enlarged images of(I-L), respectively.

FIGS. 10A and 10B show that DBC-1 is required for endocrine resistantbreast cancer cell survival. MCF-7, LCC1, and LCC9 cells cultured inhormone-free medium were electroporated with control (siCNTL) orDBC-1-specific (siDBC-1) siRNAs (21 nM) as indicated seventy-two hoursprior to harvest. In FIG. 10A, harvested cells were stained with AnnexinV-FITC and propidium iodide prior to quantification of apoptosis by flowcytometric analysis. Error bars represent the SD from the average of atleast three independent experiments performed in triplicate. In FIG.10B, cell lysates from representative apoptosis assays were resolved bySDS-10%-PAGE and processed by immunoblot analysis with antibodiesspecific for DBC-1, ERα or TFIIEβ (loading control) to validate DBC-1knockdown.

FIGS. 11A and 11B show that the DBC-1/ERα complex is resistant totamoxifen-mediated disruption in tamoxifen-resistant LCC9 cells. MCF-7,LCC1, or LCC9 cells cultured in hormone-free medium were treated withincreasing concentrations of 4-hydroxytamoxifen (4-OHT) as indicated forone hour prior to cell harvest and immunoprecipitation of whole celllysates with antibodies specific for DBC-1. Immunoprecipitates wereresolved by SDS-7.5%-PAGE and processed for immunoblot analysis withantibodies specific for DBC-1 or ERα as indicated.

DETAILED DESCRIPTION

Certain cancers, such as prostate, ovarian, endometrial and breastcancer, can be treated by hormone therapy (also called endocrinetherapy), i.e. with hormones or anti-hormone drugs that slow or stop thegrowth of certain cancers by blocking the body's natural hormones.Hormone therapy is used synonymously herein with endocrine therapy. Suchcancers may develop resistance, or be intrinsically resistant, tohormone therapy. The present application provides methods for thediagnosis and treatment of hormone-resistant or hormone-refractorycancers. As used herein the term resistance includes inherent resistance(de novo resistance) and resistance acquired during treatment (acquiredresistance) either to hormones or to anti-hormone drugs.

In breast cancer, the emergence of endocrine resistance is coincidentwith a shift from ligand-dependent to ligand-independent control ofERα-regulated breast cancer cell growth and survival. As describedherein, the deleted in breast cancer-1 gene product, DBC-1 (KIAA1967)was identified to be a direct ligand-independent binding partner of ERα.

The gene encoding DBC-1 was originally identified during a geneticsearch for candidate breast tumor suppressor genes on a human chromosome8p21 region frequently deleted in breast cancers. Refined deletionanalysis within this region revealed a second gene, deleted in breastcancer 2 (DBC-2), to encode a likely breast tumor suppressor, andconfirmed that DBC-1 expression is not substantially extinguished incancers from any source (Hamaguchi et al., PNAS 99:13647-52 (2002)). Asearch of the Oncomine database of published cancer microarray data(www.Oncomine.org), which currently permits analysis of gene expressiondata derived from 132 DNA microarray datasets among 24 different cancertypes, reveals DBC-1 to be statistically significantly upregulated inbreast carcinoma versus normal breast tissue as well as breast ductalcarcinoma versus other cancers (Radvanyi et al., PNAS 102:11005-10(2005); Biiner, Nat. Biotechnol. 23:183-4 (2005)). DBC-1 was also foundin three independent studies totaling 369 breast tumor samples to bestatistically significantly overexpressed in ER-positive versusER-negative breast tumors (van de Vijver et al., N. Engl. J. Med.347:1999-2009 (2002); Zhao et al., Mol. Biol. Cell 15:2523-36 (2004);Richardson et al., Cancer Cell 9:121-32 (2006)).

DBC-1 has been linked physically to the TNF-α/NFκB pathway by proteomicanalysis (Bouwmeester et al., Nat. Cell Biol. 6:97-105 (2004)), whilecaspase-dependent processing of DBC-1 early in apoptosis induced bydiverse stimuli, including TNF-α, was shown to unmask a proapoptoticfunction for the DBC-1 carboxyl terminus in the cytosol of moribundcells (Sundararajan et al., Oncogene 11:11 (2005)). Full-length DBC-1 ispredominantly localized to the nucleus of healthy cells, and its normalbiological function therein has heretofore remained unknown.

The present application describes that DBC-1 is a ligand independentERα-interacting protein and that DBC-1 depletion reduced thesteady-state level of unliganded ERα protein. DBC-1 amino terminus bindsdirectly to the ERα hormone-binding domain both in vitro and in vivo ina strict E2-independent manner. Furthermore, DBC-1 depletion triggeredapoptosis in cancer cells in the absence of hormone. E2-mediateddisruption of the interaction between DBC-1 and unliganded ERα abrogatedthe increase in MCF-7 cell apoptosis observed to accompany DBC-1knockdown, showing that DBC-1-bound ERα functions to suppresshormone-independent apoptosis. Therefore, ERα bound by DBC-1 promotesbreast cancer cell growth and survival in the absence of hormone. Thesefindings, described in detail in the Examples below, establish thatDBC-1 modulates ERα expression and survival activity and identifiesDBC-1 as a endocrine response determinant and therapeutic target inbreast cancer.

Provided herein is a method of determining whether a cancer cell issensitive to endocrine therapy comprising obtaining a population ofcancer cells and determining the level of expression or activity ofDBC-1 in the cells. An increase in expression or activity of DBC-1 ascompared to a control indicates that the cancer cells are not sensitiveto endocrine therapy. A decrease in expression or activity of DBC-1 ascompared to control indicates that the cancer cells are sensitive toendocrine therapy.

Also provided is a method of determining whether a cancer cell issensitive to endocrine therapy comprising obtaining a population ofcancer cells and determining the presence of binding of DBC-1 and ERα.The presence of binding of DBC-1 and ERα as compared to a controlindicates that the cancer cells are not sensitive to endocrine therapy.The absence of binding of DBC-1 and ERα indicates that the cancer cellsare sensitive to endocrine therapy.

Provided herein is a method of determining whether a subject with canceris suitable for treatment with endocrine therapy comprising obtaining abiological sample comprising cancer cells from the subject anddetermining the level of expression or activity of DBC-1. An increase inexpression or activity of DBC-1 as compared to a control indicates thatthe subject is not suitable for treatment with endocrine therapy. Adecrease in expression or activity of DBC-1 as compared to a controlindicates that the subject is suitable for treatment with endocrinetherapy.

Also provided herein is a method of determining whether a subject withcancer is suitable for treatment with endocrine therapy comprisingobtaining a biological sample comprising cancer cells from the subjectand determining the presence of binding of DBC-1 and ERα. The presenceof binding of DBC-1 and ERα as compared to a control indicates that thesubject is not suitable for treatment with endocrine therapy. Theabsence of binding of DBC-1 and ERα as compared to a control indicatesthat the subject is suitable for treatment with endocrine therapy.

Provided herein is a method of determining a susceptibility to hormoneresistant cancer in a subject comprising obtaining a biological samplecomprising cancer cells from the subject and determining the level ofexpression or activity of DBC-1. An increase in expression or activityof DBC-1 as compared to a control indicates that the subject issusceptible to hormone resistant cancer. A decrease in expression oractivity of DBC-1 as compared to a control indicates that the subject isnot susceptible to hormone resistant cancer.

Also provided is a method of determining a susceptibility to hormoneresistant cancer in a subject comprising obtaining a biological samplecomprising cancer cells from the subject and determining the presence ofbinding of DBC-1 and ERα. The presence of binding of DBC-1 and ERα ascompared to a control indicates that the subject is susceptible tohormone resistant cancer. The absence of binding of DBC-1 and ERα ascompared to a control indicates that the subject is not susceptible tohormone resistant cancer.

A method of inducing apoptosis of cancer cells is provided comprisingselecting a population of hormone resistant cancer cells and contactingthe hormone resistant cancer cells with an agent that inhibitsexpression of DBC-1. Also provided is a method of inducing apoptosis ofcancer cells comprising selecting a population of hormone resistantcancer cells and contacting the hormone resistant cancer cells with anagent that inhibits binding of DBC-1 and ERα.

A method of treating hormone resistant cancer in a subject is providedcomprising selecting a subject with hormone resistant cancer andadministering an agent that inhibits expression of DBC-1 to the subject.Also provided is a method of treating hormone resistant cancer in asubject comprising selecting a subject with hormone resistant cancer andadministering an agent that inhibits binding of DBC-1 and ERα to thesubject.

A method of reducing susceptibility of acquiring hormone resistantcancer in a subject is provided comprising selecting a subject withhormone sensitive cancer and administering an agent that inhibitsexpression of DBC-1 to the subject.

Preferably, the agents used in the provided methods are administered inan effective amount to induce apoptosis of the hormone resistant cancercells. The agent is also preferably comprised within a compositioncomprising the agent and a pharmaceutically acceptable carrier. Theinhibitors of DBC-1 expression or DBC-1/ERα binding used in the providedmethods can be, but are not limited to, a variety of functional nucleicacids, antibodies, proteins, and small molecules. The inhibitor can be,for example, an inhibitory peptide or an inhibitory nucleic acid. Theinhibitory nucleic acid can be, but is not limited to, an siRNA. Theinhibitory peptide optionally binds to the ERα binding domain of DBC-1.Inhibitors of DBC-1 expression or DBC-1/ERα binding can be furthercombined with other therapies, such as chemotherapy and/or radiotherapy.The subject of the provided methods can have endometrial cancer, breastcancer, ovarian cancer, prostate cancer, hormone sensitive cancer orhormone independent cancer.

The cancer cells used in the provided methods express one or morehormone receptors such as, for example, progesterone receptor, androgenreceptor and estrogen receptor. The term presence of binding refers tothe detection of binding of DBC-1 and ERα in hormone resistant cancercells as compared to the absence of binding in hormone sensitive cancercells or normal cells. The increase in expression of DBC-1 can bedetected by measuring DBC-1 mRNA or protein using methods well known tothose of skill in the art such as, for example, Northern and Westernblots. DBC-1 can also be detected by immunochemical methods such as, forexample, immunoblots and immunohistochemical staining, using an antibodythat binds DBC-1 or ERα. Provided herein is an antibody (monoclonal orpolyclonal) that binds DBC-1 in the region of amino acids 475-923.

The terms higher, increases, elevates, or elevation refer to increasesabove a control. For example, a control level can be the level ofexpression or activity in the same cell prior to or after recovery froma stimulus, or the control level can be the level in a control cell orpopulation of cells in the absence of a stimulus.

As used herein the term cancer cells includes all cancer cells such as,for example, hormone resistant cancer cells, hormone sensitive cancercells, endometrial cancer cells, breast cancer cells, ovarian cancercells and prostate cancer cells. The breast, prostate, endometrial orovarian cancer cells can be hormone sensitive or hormone resistant.

As used herein the term hormone resistant cancer cells includes cancercells that are inherently resistant (de novo resistance) and cancercells that have acquired resistance during treatment (acquiredresistance). Preferably, the hormone resistant cancer cells express oneor more hormone receptors. As used herein the term hormone receptorsrefers to a protein on the surface of a cell that binds to a specifichormone. Such receptors include, but are not limited to, progesteronereceptor (PR), androgen receptor (AR) and estrogen receptor (ER).

Proteins, peptides or polypeptides can be used to inhibit DBC-1expression or DBC-1/ERα binding. The term peptide, polypeptide, proteinor peptide portion is used broadly herein to mean two or more aminoacids linked by a peptide bond. The term fragment is used herein torefer to a portion of a full-length polypeptide or protein. It should berecognized that the term polypeptide is not used herein to suggest aparticular size or number of amino acids comprising the molecule andthat a peptide of the invention can contain up to several amino acidresidues or more. Preferably, the peptide that inhibits DBC-1/ERαbinding binds to the ERα binding domain of DBC-1. As used herein theterm ERα binding domain includes amino acids 1 to 150 of DBC-1. Thus,the peptide can bind DBC-1 in the region of amino acids 1 to 150 ofDBC-1.

Peptides that can be used to inhibit DBC-1/ERα binding include aminoacids 1-595 of ERα or amino acids 302-595 of ERα or fragments andvariants thereof. Thus, the peptide can begin with any amino acid from 1to 590 and end with amino acid 595. The peptide can also begin with anyamino acid from 302 to 590 and end with amino acid 595 of ERα. The aminoacid and nucleic acid sequences of ERα can be found at GenBank AccessionNos. NP_(—)000116.2 and NM_(—)000125.2, respectively. Fragments,variants, or isoforms of the ERα peptides are provided includingfunctional variants as long as the fragments, variants, functionalvariants and isoforms inhibit DBC-1/ERα binding. Peptides can be testedfor their ability to inhibit DBC-1/ERα binding by methods known to thoseof skill in the art, such as, for example, immunoassays, and the assaymethods provided herein.

It is understood that the nucleic acids that can encode theaforementioned peptide sequences, variants and fragments thereof arealso disclosed. This would include all degenerate sequences related to aspecific protein sequence, i.e. all nucleic acids having a sequence thatencodes one particular protein sequence as well as all nucleic acids,including degenerate nucleic acids, encoding the disclosed variants andderivatives of the protein sequences. Thus, while each particularnucleic acid sequence may not be written out herein, it is understoodthat each and every sequence is in fact disclosed and described hereinthrough the disclosed protein sequence. Those skilled in the art willunderstand that a wide variety of expression systems may be used toproduce ERα peptides as well as fragments, isoforms, and variants. Suchpeptides are selected based on their ability to inhibit DBC-1/ERαbinding.

Proteins that inhibit DBC-1 expression or DBC-1/ERα binding also includeantibodies with antagonistic or inhibitory properties. Such antibodiesare selected from antibodies that bind the receptor itself or antibodiesthat bind a ligand of the receptor. In addition to intact immunoglobulinmolecules, fragments, chimeras, or polymers of immunoglobulin moleculesare also useful in the methods taught herein, as long as they are chosenfor their ability to bind DBC-1, inhibit DBC-1 expression or DBC-1/ERαbinding. The antibodies can be tested for their desired activity usingin vitro assays, or by analogous methods, after which their in vivotherapeutic or prophylactic activities are tested according to knownclinical testing methods. Antibodies to DBC-1 can also be used in theprovided diagnostic methods for determining the level or activity ofDBC-1 protein.

The term antibody is used herein in a broad sense and includes bothpolyclonal and monoclonal antibodies. Monoclonal antibodies can be madeusing any procedure that produces monoclonal antibodies. For example,disclosed monoclonal antibodies can be prepared using hybridoma methods,such as those described by Kohler and Milstein, Nature, 256:495 (1975).In a hybridoma method, a mouse or other appropriate host animal istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes may beimmunized in vitro. The monoclonal antibodies may also be made byrecombinant DNA methods, such as those described in U.S. Pat. No.4,816,567 (Cabilly et al.). DNA encoding the disclosed monoclonalantibodies can be readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains ofmurine antibodies). Libraries of antibodies or active antibody fragmentscan also be generated and screened using phage display techniques, e.g.,as described in U.S. Pat. No. 5,804,440 to Burton et al. and U.S. Pat.No. 6,096,441 to Barbas et al.

Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 published Dec. 22, 1994and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typicallyproduces two identical antigen binding fragments, called Fab fragments,each with a single antigen binding site, and a residual Fc fragment.Pepsin treatment yields a fragment that has two antigen combining sitesand is still capable of cross linking antigen.

The antibody fragments, whether attached to other sequences or not, canalso include insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the antibody or antibody fragment is notsignificantly altered or impaired compared to the non-modified antibodyor antibody fragment. These modifications can provide for someadditional property, such as to remove/add amino acids capable ofdisulfide bonding, to increase its bio-longevity, to alter its secretorycharacteristics, etc. In any case, the antibody or antibody fragmentmust possess a bioactive property, such as specific binding to itscognate antigen. Functional or active regions of the antibody orantibody fragment may be identified by mutagenesis of a specific regionof the protein, followed by expression and testing of the expressedpolypeptide. Such methods are readily apparent to a skilled practitionerin the art and can include site-specific mutagenesis of the nucleic acidencoding the antibody or antibody fragment. (Zoller, M. J. Curr. Opin.Biotechnol. 3:348-354, 1992).

As used herein, the term antibody or antibodies can also refer to ahuman antibody and/or a humanized antibody. Examples of techniques forhuman monoclonal antibody production include those described by Cole etal. (Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77,1985) and by Boerner et al. (J. Immunol., 147(1): δ 95, 1991). Humanantibodies (and fragments thereof) can also be produced using phagedisplay libraries (Hoogenboom et al., J. Mol. Biol., 227:381, 1991;Marks et al., J. Mol. Biol., 222:581, 1991). The disclosed humanantibodies can also be obtained from transgenic animals. For example,transgenic, mutant mice that are capable of producing a full repertoireof human antibodies, in response to immunization, have been described(see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 255(1993); Jakobovits et al., Nature, 362:255 258 (1993); Bruggermann etal., Year in Immunol., 7:33 (1993)). Specifically, the homozygousdeletion of the antibody heavy chain joining region (J(H)) gene in thesechimeric and germ line mutant mice results in complete inhibition ofendogenous antibody production, and the successful transfer of the humangerm line antibody gene array into such germ line mutant mice results inthe production of human antibodies upon antigen challenge.

Antibody humanization techniques generally involve the use ofrecombinant DNA technology to manipulate the DNA sequence encoding oneor more polypeptide chains of an antibody molecule. Accordingly, ahumanized form of a non human antibody (or a fragment thereof) is achimeric antibody or antibody chain that contains a portion of anantigen binding site from a non-human (donor) antibody integrated intothe framework of a human (recipient) antibody. Fragments of humanizedantibodies are also useful in the methods taught herein. As usedthroughout, antibody fragments include Fv, Fab, Fab′, or other antigenbinding portion of an antibody. Methods for humanizing non humanantibodies are well known in the art. For example, humanized antibodiescan be generated according to the methods of Winter and co workers(Jones et al., Nature, 321:522 525 (1986), Riechmann et al., Nature,332:323 327 (1988), Verhoeyen et al., Science, 239:1534 1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Methods that can be used to producehumanized antibodies are also described in U.S. Pat. No. 4,816,567(Cabilly et al.), U.S. Pat. No. 5,565,332 (Hoogenboom et al.), U.S. Pat.No. 5,721,367 (Kay et al.), U.S. Pat. No. 5,837,243 (Deo et al.), U.S.Pat. No. 5,939,598 (Kucherlapati et al.), U.S. Pat. No. 6,130,364(Jakobovits et al.), and U.S. Pat. No. 6,180,377 (Morgan et al.).

Also provided herein are functional nucleic acids that inhibitexpression of DBC-1. Such functional nucleic acids include but are notlimited to antisense molecules, aptamers, ribozymes, triplex formingmolecules, RNA interference (RNAi), and external guide sequences. Thus,for example, a small interfering RNA (siRNA) could be used to reduce oreliminate expression of DBC-1.

Functional nucleic acids are nucleic acid molecules that have a specificfunction, such as binding a target molecule or catalyzing a specificreaction. Functional nucleic acid molecules can interact with anymacromolecule, such as DNA, RNA, polypeptides, or carbohydrate chains.Thus, functional nucleic acids can interact with the mRNA, genomic DNA,or polypeptide. Often functional nucleic acids are designed to interactwith other nucleic acids based on sequence homology between the targetmolecule and the functional nucleic acid molecule. In other situations,the specific recognition between the functional nucleic acid moleculeand the target molecule is not based on sequence homology between thefunctional nucleic acid molecule and the target molecule, but rather isbased on the formation of tertiary structure that allows specificrecognition to take place.

Antisense molecules are designed to interact with a target nucleic acidmolecule through either canonical or non-canonical base pairing. Theinteraction of the antisense molecule and the target molecule isdesigned to promote the destruction of the target molecule through, forexample, RNAseH mediated RNA-DNA hybrid degradation. Alternatively theantisense molecule is designed to interrupt a processing function thatnormally would take place on the target molecule, such as transcriptionor replication. Antisense molecules can be designed based on thesequence of the target molecule. Numerous methods for optimization ofantisense efficiency by finding the most accessible regions of thetarget molecule exist. Exemplary methods would be in vitro selectionexperiments and DNA modification studies using DMS and DEPC.

Aptamers are molecules that interact with a target molecule, preferablyin a specific way. Typically aptamers are small nucleic acids rangingfrom 15-50 bases in length that fold into defined secondary and tertiarystructures, such as stem-loops or G-quartets. Representative examples ofhow to make and use aptamers to bind a variety of different targetmolecules can be found in, for example, U.S. Pat. Nos. 5,476,766 and6,051,698.

Ribozymes are nucleic acid molecules that are capable of catalyzing achemical reaction, either intramolecularly or intermolecularly. Thereare a number of different types of ribozymes that catalyze nuclease ornucleic acid polymerase type reactions which are based on ribozymesfound in natural systems, such as hammerhead ribozymes, hairpinribozymes and tetrahymena ribozymes). There are also a number ofribozymes that are not found in natural systems, but which have beenengineered to catalyze specific reactions de novo (for example, but notlimited to U.S. Pat. Nos. 5,807,718, and 5,910,408). Ribozymes maycleave RNA or DNA substrates. Representative examples of how to make anduse ribozymes to catalyze a variety of different reactions can be foundin U.S. Pat. Nos. 5,837,855, 5,877,022, 5,972,704, 5,989,906, and6,017,756.

Triplex forming functional nucleic acid molecules are molecules that caninteract with either double-stranded or single-stranded nucleic acid.When triplex molecules interact with a target region, a structure calleda triplex is formed, in which there are three strands of DNA forming acomplex dependant on both Watson-Crick and Hoogsteen base-pairing.Triplex molecules are preferred because they can bind target regionswith high affinity and specificity. Representative examples of how tomake and use triplex forming molecules to bind a variety of differenttarget molecules can be found in U.S. Pat. Nos. 5,650,316, 5,683,874,5,693,773, 5,834,185, 5,869,246, 5,874,566, and 5,962,426.

External guide sequences (EGSs) are molecules that bind a target nucleicacid molecule forming a complex, and this complex is recognized by RNaseP, which cleaves the target molecule. EGSs can be designed tospecifically target a RNA molecule of choice. Representative examples ofhow to make and use EGS molecules to facilitate cleavage of a variety ofdifferent target molecules be found in U.S. Pat. Nos. 5,168,053,5,624,824, 5,683,873, 5,728,521, 5,869,248, and 5,877,162.

Gene expression can also be effectively silenced in a highly specificmanner through RNA interference (RNAi). Short Interfering RNA (siRNA) isa double-stranded RNA that can induce sequence-specificpost-transcriptional gene silencing, thereby decreasing or eveninhibiting gene expression. In one example, an siRNA triggers thespecific degradation of homologous RNA molecules, such as mRNAs, withinthe region of sequence identity between both the siRNA and the targetRNA. Sequence specific gene silencing can be achieved in mammalian cellsusing synthetic, short double-stranded RNAs that mimic the siRNAsproduced by the enzyme dicer. siRNA can be chemically or invitro-synthesized or can be the result of short double-strandedhairpin-like RNAs (shRNAs) that are processed into siRNAs inside thecell. Synthetic siRNAs are generally designed using algorithms and aconventional DNA/RNA synthesizer. Suppliers include Ambion (Austin,Tex.), ChemGenes (Ashland, Mass.), Dharmacon (Lafayette, Colo.), GlenResearch (Sterling, Va.), MWB Biotech (Esbersberg, Germany), Proligo(Boulder, Colo.), and Qiagen (Vento, The Netherlands). siRNA can also besynthesized in vitro using kits such as Ambion's SILENCER® siRNAConstruction Kit (Ambion, Austin, Tex.).

Methods of screening for agents that inhibit expression of DBC-1 areprovided. Such a screening method comprises the steps of providing acell that expresses DBC-1; contacting the cell with a candidate agent tobe tested; and quantifying the expression of DBC-1. The contacting stepcan be in vitro.

Methods of screening for agents that inhibit DBC-1/ERα binding are alsoprovided. Such a screening method comprises the steps of providing acell that expresses ERα and DBC-1, wherein DBC-1 and ERα bind within thecell; contacting the cell with a candidate agent to be tested; anddetermining whether the candidate agent disrupts or prevents binding ofDBC-1 and ERα. Another method of screening for agents that inhibitDBC-1/ERα binding comprises the steps of providing a sample comprisingERα and DBC-1, wherein DBC-1 is capable of binding ERα in the sample;contacting the sample with a candidate agent to be tested; anddetermining whether the candidate agent disrupts binding of DBC-1 andERα.

Such methods allow one skilled in the art to select candidate agentsthat exert a regulating effect on the expression level of DBC-1 or thebinding of DBC-1 and ERα. Such agents may be useful as activeingredients included in pharmaceutical compositions for treatingpatients suffering from cancer. The cell in the methods above can be acell that normally expresses DBC-1 and/or ERα. The cell can be a hormoneresistant cancer cell that expresses DBC-1 and ERα. The cell can also bea prokaryotic or an eukaryotic cell that has been transfected with anucleotide sequence encoding DBC-1 and/or ERα or a variant or a fragmentthereof, operably linked to a promoter. Using DNA recombinationtechniques well known by the one skill in the art, protein encoding DNAsequences can be inserted into an expression vector, downstream from apromoter sequence.

Quantification of expression of DBC-1 may be realized either at the mRNAlevel or at the protein level. In the latter case, antibodies may beused to quantify the amounts of DBC-1 protein, for example in an ELISAor a RIA assay. Quantification of DBC-1 mRNA may be realized by aquantitative PCR amplification of the cDNA obtained by a reversetranscription of the total mRNA of the cell expressing DBC-1, using apair of primers specific for DBC-1.

Methods for determining whether the candidate agent disrupts binding ofDBC-1 and ERα are well known to those of skill in the art. The assay canbe, for example, an immunoassay, radioimmunoassay (RIA), enzyme-linkedimmunosorbent assay (ELISA) and a non-competitive binding assay.Competitive binding assays wherein an unlabeled and a labeled analytecompete for sites to bind to a specific protein can also be used.

Pharmaceutical compositions comprising one or more of the inhibitors oragents provided herein may include carriers, thickeners, diluents,buffers, preservatives, surface active agents and the like in additionto the molecule of choice. Pharmaceutical compositions may also includeone or more active ingredients such as antimicrobial agent, achemotherapeutic agent, and the like. The compositions of the presentapplication can be administered in vivo in a pharmaceutically acceptablecarrier. By pharmaceutically acceptable is meant a material that is notbiologically or otherwise undesirable. Thus, the material may beadministered to a subject, without causing undesirable biologicaleffects or interacting in a deleterious manner with any of the othercomponents of the pharmaceutical composition in which it is contained.The carrier would naturally be selected to minimize any degradation ofthe active ingredient and to minimize any adverse side effects in thesubject, as would be well known to one of skill in the art.

The disclosed compositions can be administered in a number of waysdepending on whether local or systemic treatment is desired, and on thearea to be treated. Thus, the disclosed compositions can beadministered, for example, orally, parenterally (e.g., intravenously),by intramuscular injection, by intraperitoneal injection, transdermally,extracorporeally, or topically.

The materials may be in solution, suspension (for example, incorporatedinto microparticles, liposomes, or cells). These may be targeted to aparticular cell type via antibodies, receptors, or receptor ligands.Suitable carriers and their formulations are described in Remington: TheScience and Practice of Pharmacy (21th ed.) ed. David B. Troy,Lippincott Williams & Wilkins, 2005. Typically, an appropriate amount ofa pharmaceutically-acceptable salt is used in the formulation to renderthe formulation isotonic. Examples of the pharmaceutically-acceptablecarrier include, but are not limited to, saline, Ringer's solution anddextrose solution. The pH of the solution is preferably from about 5 toabout 8.5, and more preferably from about 7.8 to about 8.2. Furthercarriers include sustained release preparations such as semipermeablematrices of solid hydrophobic polymers, which matrices are in the formof shaped articles, e.g., films, liposomes or microparticles. It will beapparent to those persons skilled in the art that certain carriers maybe more preferable depending upon, for instance, the route ofadministration and concentration of composition being administered.

The terms effective amount and effective dosage are usedinterchangeably. The term effective amount is defined as any amountnecessary to produce a desired physiologic response. Effective amountsand schedules for administering the compositions may be determinedempirically, and making such determinations is within the skill in theart. The dosage ranges for the administration of the compositions arethose large enough to produce the desired effect in which the symptomsor disorder are affected. The dosage should not be so large as to causesubstantial adverse side effects, such as unwanted cross-reactions,anaphylactic reactions, and the like. Generally, the dosage will varywith the age, condition, sex, type of disease and extent of the diseasein the patient, route of administration, or whether other drugs areincluded in the regimen, and can be determined by one of skill in theart. The dosage can be adjusted by the individual physician in the eventof any contraindications. Dosage can vary, and can be administered inone or more dose administrations daily, for one or several days.Guidance can be found in the literature for appropriate dosages forgiven classes of pharmaceutical products.

The inhibitor of DBC-1 expression or DBC-1/ERα binding can beadministered in combination with one or more other therapeutic orprophylactic regimens, such as, for example, chemotherapy. As usedthroughout, a therapeutic agent is a compound or composition effectivein ameliorating a pathological condition. Illustrative examples oftherapeutic agents include, but are not limited to, an anti-cancercompound, anti-inflammatory agents, anti-viral agents, anti-retroviralagents, anti-opportunistic agents, antibiotics, immunosuppressiveagents, immunoglobulins, and antimalarial agents.

An anti-cancer compound or chemotherapeutic agent is a compound orcomposition effective in inhibiting or arresting the growth of anabnormally growing cell. Thus, such an agent may be used therapeuticallyto treat cancer as well as other diseases marked by abnormal cellgrowth. A pharmaceutically effective amount of an anti-cancer compoundis an amount administered to an individual sufficient to causeinhibition or arrest of the growth of an abnormally growing cell.Illustrative examples of anti-cancer compounds include: bleomycin,carboplatin, chlorambucil, cisplatin, colchicine, cyclophosphamide,daunorubicin, dactinomycin, diethylstilbestrol doxorubicin, etoposide,5-fluorouracil, floxuridine, melphalan, methotrexate, mitomycin,6-mercaptopurine, teniposide, 6-thioguanine, vincristine andvinblastine.

Inhibitors of DBC-1 expression or DBC-1/ERα binding can be furthercombined with other therapies, such as chemotherapy and/or radiotherapyin the treatment of malignancy, and therapeutic efficacy can be enhancedby apoptosis-inducing compounds.

Any of the aforementioned treatments can be used in any combination withthe inhibitors described herein. Thus, for example, the inhibitors canbe administered in combination with a chemotherapeutic agent andradiation. Other combinations can be administered as desired by those ofskill in the art. Combinations may be administered either concomitantly(e.g., as an admixture), separately but simultaneously (e.g., viaseparate intravenous lines into the same subject), or sequentially(e.g., one of the compounds or agents is given first followed by thesecond). Thus, the term combination is used to refer to eitherconcomitant, simultaneous, or sequential administration of two or moreagents.

There are a variety of sequences related to, for example, DBC-1 and ERαthat are disclosed on Genbank, at www.pubmed.gov and these sequences andothers are herein incorporated by reference in their entireties as wellas for individual subsequences contained therein. For example, the aminoacid and nucleic acid sequences of DBC-1 (KIAA1967) can be found atGenBank Accession Nos. NP_(—)066997.3 and NM_(—)021174.4, respectively.The amino acid and nucleic acid sequences of ERα can be found at GenBankAccession Nos. NP_(—)000116.2 and NM_(—)000125.2, respectively.

The term isolated requires that the material be removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring).

As used throughout, by a subject is meant an individual. Thus, thesubject can include domesticated animals, such as cats, dogs, etc.,livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratoryanimals (e.g., mouse, rabbit, rat, guinea pig, etc.) and birds.Preferably, the subject is a mammal such as a primate, and, morepreferably, a human.

Ranges may be expressed herein as from about one particular value,and/or to about another particular value. Similarly, when values areexpressed as approximations, by use of the term about, it will beunderstood that the particular value is included. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint.

The absence of binding as used herein refers to a level of binding thatis at or below detectable levels using detection methods known in theart and described herein. The absence of binding includes a level ofbinding that is about less that 1.5 times above background usingdetection methods. The presence of binding refers to a level of bindingthat is detectable and includes, for example, a level of binding that isabout 1.5 times or greater above background levels using detectionmethods.

Inhibit, inhibiting, and inhibition mean to decrease an activity,response, condition, disease, or other biological parameter. This caninclude but is not limited to the complete ablation of the activity,response, condition, or disease. This may also include, for example, a10% reduction in the activity, response, condition, or disease ascompared to the native or control level. Thus, the reduction can be a10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction inbetween as compared to native or control levels.

As used herein the terms treatment, treat or treating refers to a methodof reducing the effects of a disease or condition or at least onesymptom of the disease or condition. Thus in the disclosed methodtreatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or100% reduction in the severity of an established disease or condition orsymptom of the disease or condition. For example, the method fortreating cancer is considered to be a treatment if there is a 10%reduction in one or more symptoms of the disease in a subject ascompared to control. Thus the reduction can be a 10, 20, 30, 40, 50, 60,70, 80, 90, 100% or any percent reduction in between 10 and 100 ascompared to native or control levels. It is understood that treatmentdoes not necessarily refer to a cure or complete ablation of thedisease, condition or symptoms of the disease or condition.

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed method and compositions. These and othermaterials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed that while specific reference of each various individual andcollective combinations and permutation of these compounds may not beexplicitly disclosed, each is specifically contemplated and describedherein. For example, if an inhibitor is disclosed and discussed and anumber of modifications that can be made to a number of molecules of theinhibitor are discussed, each and every combination and permutation ofinhibitor and the modifications that are possible are specificallycontemplated unless specifically indicated to the contrary. This conceptapplies to all aspects of this application including, but not limitedto, steps in methods of making and using the disclosed compositions.Thus, if there are a variety of additional steps that can be performedit is understood that each of these additional steps can be performedwith any specific embodiment or combination of embodiments of thedisclosed methods, and that each such combination is specificallycontemplated and should be considered disclosed.

Publications cited herein and the material for which they are cited arehereby specifically incorporated by reference. No admission is made thatany reference constitutes prior art. The discussion of references stateswhat their authors assert, and applicants reserve the right to challengethe accuracy and pertinency of the cited documents.

EXAMPLES Example 1 Disruption of DBC-1/ER-α Binding Promotes Apoptosisof Cancer Cells

Materials and Methods

Expression Plasmids. pCS2+-ERα was constructed by subcloning aBamHI-BamHI fragment carrying the full-length coding region of ERα cDNAfrom pG/ER(G) (Liu and Picard, FEMS Microbiol. Lett. 159:167-71 (1998))into pCS2+ (Turner and Weintraub, Genes Dev. 8:1434-47 (1994)). pACT-ERαwas constructed by subcloning a BamHI-BamHI fragment carrying thefull-length coding region of ERα cDNA from pCS2+-ERα into the pACT VP16fusion vector (Promega Corporation, Madison, Wis.). GST-ERα (1-184),GST-ERα (185-250), GST-ERα (251-301), and GST-ERα (302-595) weredescribed previously (Qi et al., J. Biol. Chem. 277:28624-30 (2002)).GST-ERα (1-595) was generated by amplifying ERα by PCR and inserting itinto the EcoRI site of pGEX-4T-3 vector (Amersham Biosciences,Piscataway, N.J.).

pSport1-DBC-1 was a clone obtained from RZPD German Resource Center forGenome Research GmbH (Berlin, Germany) (RZPD clone ID: DKFZp761O0817Q;KIAA1967). pCS2+DBC-1 was constructed by first amplifying the aminoterminal half of DBC-1 by PCR and inserting it into the ClaI/EcoRI siteof pCS2+.His6.FLAG, which yielded pCS2+.His6.FLAG-5′DBC-1.SphI. Thecarboxyl terminal half of DBC-1 was amplified by PCR and then insertedinto the SphI/EcoRI site of pCS2+.His6.FLAG-5′DBC-1.SphI to yieldpCS2+DBC-1, which contains a STOP codon between the DBC-1 codingsequence and the His6.FLAG fusion. This construct was confirmed bysequencing. pCS2+.His6.FLAG-DBC-1 was generated by amplifying thecarboxyl terminal half of DBC-1 by PCR and then inserting it into theSphI/EcoRI site of pCS2+.His6.FLAG-5′DBC-1.SphI to create a version ofDBC-1 fused to C-terminal 6×His and FLAG tags. pCS2+-DBC-1amino-terminal fragments (1-478, 1-300, 1-230, 1-200, 1-150, and150-478) were generated by amplifying fragments by PCR and insertingthem into the EcoRI/XhoI site of pCS2+. pCS2+-DBC-1 (479-923) wasgenerated by amplifying the carboxyl terminal half of DBC-1 by PCR andinserting it into the EcoRI/XhoI site of pCS2+. pBIND-DBC1 (1-478) wasconstructed by amplifying the amino terminal half of DBC-1 by PCR andinserting it into the SalI/XbaI site of the pBIND GAL4 fusion vector(Promega Corporation, Madison, Wis.). pBINDDBC1 (479-923) wasconstructed by amplifying the carboxyl terminal half of DBC-1 by PCR andinserting it into the XbaI/NotI site of pBIND. pBIND-DBC-1 wasconstructed by subcloning an XbaI/NotI carboxyl terminal fragment ofDBC-1 from pBIND-DBC1 (479-923) into pBIND-DBC1 (1-478).

Reporter Plasmids. pG5luc, carrying five GAL4 DNA-binding sites upstreamof the major late promoter of adenovirus driving expression of thefirefly luciferase gene, was purchased from Promega Corporation,Madison, Wis.

Cell Lines and Culture Conditions. The HeLa (ATCC, Manassas, Va.), T-47D(ATCC, Manassas, Va.), MCF-7 (ATCC, Manassas, Va.), AmphoPack 293(Clontech, Mountain View, Calif.), and MDA-MB-231 (ATCC, Manassas, Va.)cells were routinely cultured in Dulbecco's modified Eagle medium (GibcoBRL, Gaithersburg, Md.) supplemented with 10% fetal bovine serum(Hyclone, Logan, Utah) and penicillin-streptomycin-L-glutamine (GibcoBRL, Gaithersburg, Md.). BG-1 cells, (Ignar-Trowbridge et al., Mol.Endocrinol. 7:992-8 (1993)), were routinely cultured in Dulbecco'smodified Eagle medium:F12 (Gibco BRL, Gaithersburg, Md.) supplemented aslisted above. All cell lines except BG-1 and MDA-MB-231 cells werecultured at 37° C. in a 10% CO2 humidified chamber; BG-1 and MDA-MB-231cells were cultured at 5% CO2.

GST Pull-down Assays. GST and GST fusion proteins were expressed in andpurified from BL21-CodonPlus(DE3)-RIPL Escherichia coli (Stratagene, LaJolla, Calif.). Cells were grown at 37° C. to A600 of 1.0, thenisopropyl-1-thio-β-D-galactopyranoside (IPTG) was added to a finalconcentration of 0.5 mM. For GST, GST-ERα (1-184), GST-ERα (185-250),and GST-ERα (251-301), the cells were grown at 30° C. for another 5hours. For GST-ERα (302-595), the cells were grown at 20° C. for another5 hours. For GST-ERα (1-595), the cells were grown at 16° C. for another5 hours. Cells were pelleted, washed once with phosphate bufferedsaline, and resuspended in lysis 250 buffer (50 mM Tris-HCl, 250 mMNaCl, 5 mM EDTA, 0.1% NP-40) supplemented with protease inhibitors (20μM antipain, 2 μM pepstatin, 20 μM leupeptin, 2 μg/ml aprotinin).Resuspended cells were subjected to one round of freeze-thaw followed bysonication and clarification by centrifugation at 35,000×g for 30 min at4° C. Clarified GST lysates were bound to glutathione-Sepharose beads(Amersham Biosciences, Piscataway, N.J.) for 45 min at 25° C., followedby washing four times for 5 min each with lysis 250 buffer containing0.2% bovine serum albumin (BSA) and protease inhibitors. DBC-1 orfragments of DBC-1 were labeled with [35S]methionine (TNT SP6 quickcoupled transcription/translation system; Promega Corporation, Madison,Wis.) and incubated with immobilized GST proteins in PD buffer (50 mMTris-HCl, 200 mM KCl, 5 mM MgCl₂, 5 mM EDTA, 0.05% NP-40) for 2 h at 4°C. Binding reactions were washed with PD buffer three times for 5 mineach at 4° C. and subsequently boiled in 20 μl of 1× Laemmli samplebuffer. Eluates were resolved by SDS-12%-PAGE and visualized byPhosphorlmager analysis (Amersham Biosciences, Piscataway, N.J.).

Mammalian Two-Hybrid Interaction Analysis. HeLa cells grown underhormone-free conditions for two days were plated at 1×10⁵ cells per wellin 12-well plates (Corning, Corning, N.Y.). After 24 hours, the cellswere transfected using FuGENE® 6 (Roche, Basel, Switzerland) accordingto the manufacturer's recommendations. In defining the ERα-DBC-1interaction, transfection mixtures consisted of pCH110 (Zheng et al.,PNAS 98:9587-92 (2001)), an internal control plasmid, expressingβ-galactosidase under control of the SV40 promoter (167 ng), pG5lucreporter (167 ng), pACT-ERα (334 ng), and the various pBIND-DBC-1constructs (334 ng), including pBIND-DBC-1, pBIND-DBC-1 (1-478), andpBIND-DBC-1 (479-923). pBIND empty vector was used as an appropriatecontrol for interaction with pACTERα pACT empty vector was used as anappropriate control for interaction with the various pBIND-DBC-1constructs. After 48 hours, cells were harvested and assayed forluciferase activity according to the manufacturer's guidelines (PromegaCorporation, Madison, Wis.). Luciferase activity was corrected for thecorresponding β-galactosidase activity to give relative activity.β-galactosidase activity was assayed according to manufacturer'sinstructions (Tropix, Bedford, Mass.). Transfections were repeated aminimum of three times in duplicate. For experiments with ligandtreatment, 17-β-estradiol (E2; Sigma-Aldrich, St. Louis, Mo.) was addedto cells at 10⁻⁷ M for 24 hours prior to harvest.

For Western blot analysis, 48 hours posttransfection, whole cell lysateswere prepared in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5%deoxycholate, 1% NP-40, 0.1% SDS) supplemented with protease inhibitorsand clarified by centrifugation. Equivalent amounts of lysates wereboiled in Laemmli sample buffer and resolved by SDS-10%-PAGE. Proteinswere analyzed by immunoblot using antibodies against GAL4-DBD (RK5C1;Santa Cruz, Santa Cruz, Calif.) and ERα (HC-20; Santa Cruz, Santa Cruz,Calif.).

Co-immunoprecipitations. T-47D, MCF-7, or BG-1 cells were grown underhormone-free conditions for three days and treated without or with17-β-estradiol (10⁻⁷ M) or 4-hydroxytamoxifen (10⁻⁶ M; Sigma-Aldrich,St. Louis, Mo.) or ICI 182,780 (10⁻⁷ M; Tocris, Ellisville, Mo.) for onehour prior to cell harvest and co-immunoprecipitation. Whole celllysates were prepared in 0.5% NP-40 lysis buffer (50 mM Tris-HCl, 150 mMNaCl, 5 mM EDTA, 0.5% NP-40) supplemented with protease inhibitors andclarified by centrifugation. Nuclear and cytoplasmic extracts wereprepared as described (Dignam et al., Nucleic Acid Res. 11:1475-89(1983)). Lysates were adjusted to binding buffer (50 mM Tris-HCl, 175 mMNaCl, 5 mM EDTA, 0.2% NP-40, 10% glycerol, supplemented with proteaseinhibitors) concentration. Lysates were then subjected toimmunoprecipitation with rabbit polyclonal anti-ERα (HC-20; Santa Cruz,Santa Cruz, Calif.) antibody or mouse polyclonal anti-DBC-1 antibody(produced in our laboratory against recombinant DBC-1 (amino acids475-923)) and protein A Sepharose beads. Immune complexes were washedthree times with binding buffer, boiled in Laemmli sample buffer, andresolved by SDS-10%-PAGE. Proteins were transferred to nitrocellulosemembranes and visualized by using antibodies against DBC-1, ERα, HSP90(rabbit polyclonal; GeneTex, San Antonio, Tex.), CYP40 (rabbitpolyclonal; AbCam, Cambridge, Mass.), appropriate peroxidase-conjugatedsecondary antibodies (Biorad, Hercules, Calif.), and enhancedchemiluminescence detection (Amersham Biosciences, Piscataway, N.J.).

DBC-1 Silencing by siRNA. To selectively knockdown the expression ofendogenous DBC-1 protein, an siRNA pool consisting for 4 differenttarget sequences was used (catalog #D-010427-01 sense sequenceCAACUGGUGUGGCUACUUGUU (SEQ ID NO:1), antisense sequence5′-PCAAGUAGCCACACCAGUUGUU (SEQ ID NO:2); catalog #D-010427-02 sensesequence CUACUGAGCCUUCCUGAAAUU (SEQ ID NO:3), antisense sequence5′-PUUUCAGGAAGGCUCAGUAGUU (SEQ ID NO:4); catalog #D-010427-03 sensesequence CAGCUUGCAUGACUACUUUUU (SEQ ID NO:5) antisense sequence5′-PAAAGUAGUCAUGCAAGCUGUU (SEQ ID NO:6); catalog #D-010427-04 sensesequence CAGCGGGUCUUCACUGGUAUU (SEQ ID NO:7) antisense sequence5′-PUACCAGUGAAGACCCGCUGUU (SEQ ID NO:8); Dharmacon, Chicago, Ill.).

These RNA duplexes (3 μg per 2×10⁶ cells), as well as a negative controlduplex that does not pair with any human mRNA (Dharmacon, Chicago,Ill.), were electroporated in MCF-7 or MDA-MB-231 cells using the cellline Nucleofector® kit V (Amaxa, Gaithersburg, Md.). Immediatelyfollowing control or DBC-1 siRNA electroporation, cells were seeded at aconcentration of 1×10⁶ per 60 mm plate. In all experiments, cells wereallowed to grow for three days in phenol-red free medium supplementedwith 10% charcoal/dextran-treated FBS and without or with indicatedchemical treatments. Cells were harvested three dayspost-electroporation.

Western Blot Analysis. Three days postelectroporation, whole celllysates were prepared in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5%deoxycholate, 1% NP-40, 0.1% SDS) supplemented with protease inhibitorsand clarified by centrifugation. Lysates were boiled in Laemmli samplebuffer and resolved by SDS-10%-PAGE. Proteins were analyzed byimmunoblot using antibodies against DBC-1 (produced in our laboratory),ERα (HC-20; Santa Cruz, Santa Cruz, Calif.), and TFIIEβ (C-21; SantaCruz, Santa Cruz, Calif.) as previously described. Quantification ofWestern blots was performed using the Kodak ImageStation 2000R.

Quantitative Real-Time RT-PCR. Three days post-electroporation, RNA wasisolated from cells using TRIZOL® reagent (Invitrogen, Carlsbad,Calif.). RNA was reverse transcribed using random hexamers andSUPERSCRIPT® III (Invitrogen, Carlsbad, Calif.) following themanufacturer's instructions. Quantitative RT-PCR was performed usingABSOLUTE™ SYBR® Green ROX Mix (ABgene, Rochester, N.Y.) on an ABI PRISM7900HT Fast real-time PCR system (Applied Biosystems, Foster City,Calif.). The gene-specific primers used were as follows: DBC-1 (5′-ATGTCC CAG TTT AAG CGC CAG-3′ (SEQ ID NO:9) and 5′-CAA CCC CAA AGT AGT CATGCA A-3′ (SEQ ID NO:10)), ERα (5′-CCA CCA ACC AGT GCA CCA TT-3′ (SEQ IDNO:11) and 5′-GGT CTT TTC GTA TCC CAC CTT TC-3′ (SEQ ID NO:12)), andGAPDH (5′-CCT GTT CGA CAG TCA GCC G-3′ (SEQ ID NO:13) and 5′-CGA CCA AATCCG TTG ACT CC-3′ (SEQ ID NO:14)).

Proliferation Assays. Three days prior to electroporation, cells weregrown in phenol-red free medium supplemented with 10%charcoal/dextran-treated FBS. Immediately following control or DBC-1siRNA electroporation, cells were seeded at a concentration of 10×10⁴per well in 6-well plates. In all experiments, triplicates of cells wereallowed to grow for seven days in phenol-red free medium supplementedwith 10% charcoal/dextran-treated FBS and without or with 17-β-estradiol(10⁻⁷ M) at 37° C. and 10% CO₂. Cell viability was determined using thetrypan blue exclusion assay, and viable cells were counted with the useof a hemacytometer. Proliferation assays were repeated a minimum ofthree times.

Apoptosis Assays. Three days prior to electroporation, cells were grownin phenol-red free medium supplemented with 10% charcoal/dextran-treatedFBS. Immediately following control or DBC-1 siRNA electroporation, cellswere seeded at a concentration of 1×10⁶ per 60 mm plate. In allexperiments, cells were allowed to grow for three days in phenol-redfree medium supplemented with 10% charcoal/dextran-treated FBS andwithout or with 17-β-estradiol (10⁻⁷ M), ICI 182,780 (10⁻⁷ M; Tocris,Ellisville, Mo.), or a combination of the two at 37° C. and 10% CO₂.Seventy-two hours postelectroporation, trypsinized cells (1×10⁵) werestained with Annexin V-FITC (BD Pharmingen, San Jose, Calif.) andpropidium iodide (Becton Dickison (BD), Franklin Lakes, N.J.) accordingto the manufacturer's instructions. Flowcytometric analyses to quantifyapoptosis were done in a FACSCALIBUR™ (BD, Franklin Lakes, N.J.). AllAnnexin V-FITC positive cells were considered apoptotic. Apoptosisassays were repeated a minimum of three times.

Data Analysis. Statistical significance was assessed by comparing meanvalues (±SD) values with Student's t-test for independent groups. p≦0.05was considered statistically significant.

Results

DBC-1 Interacts with ERα in vivo in a Ligand-Independent Manner. DBC-1was identified by mass spectrometric-based peptide sequence analysis ofproteins coimmunoprecipitated specifically with unliganded, but notliganded, ERα (FIGS. 8A, 8B and 8C). To facilitate the isolation ofERα-associated proteins, retroviral-mediated gene transfer to engineer aHeLaS3 human cervical carcinoma-derived cell line (fERalS3) that stablyexpresses a FLAG epitope-tagged ERα was employed (fERα). Initially, tocharacterize the chromatographic profile of fERα in these cells as afunction of E2, nuclear extracts derived from hormone-deprived andhormone-stimulated fERα/S3 cells were fractionated by phosphocellulose(PC-11) chromatography. Individual step fractions were then analyzed byimmunoblot analysis for the presence of fERα (FIG. 8A). This analysisrevealed a marked ligand-induced shift in the chromatographic peak offERα from a 0.3 M KCl step fraction in the absence of E2 to a 0.5 M KClstep fraction in the presence of E2 (FIG. 8A). This ligand-inducedswitch in the chromatographic profile of fERα is consistent with theexchange of fERα between ligand-independent and ligand-dependent proteinpartners.

To isolate ligand-independent ERα-associated proteins, the PC-11 0.3 MKCl step fraction from hormone deprived ERalS3 nuclear extracts waschromatographed on an anti-FLAG M2 monoclonal antibody affinity column.As a negative control, a comparable 0.3 M KCl step fraction fromhormone-deprived parental HeLa S3 nuclear extracts was subjected to FLAGmonoclonal antibody affinity chromatography (FIG. 8B). This procedureresulted in the specific isolation of fERα along with approximately 25ERα-associated proteins (ERAPs) ranging in size from approximately 30 to300 kDa (FIG. 8C). No ERAPs were recovered following M2 antibodyaffinity chromatography of partially purified nuclear extracts derivedfrom the parental HeLaS3 cell line. This result indicates that ERAPsobserved following M2 antibody affinity chromatography of partiallypurified fERα/S3 nuclear extracts were recovered by virtue of theirspecific association with FLAG epitope-tagged ERα. Mass spectrometricbased peptide sequence analysis of size-selected ERAP pools revealed oneERAP to be the product of the Deleted in Breast Cancer-1 gene, DBC-1.

To determine if DBC-1 interacts directly with unliganded ERα in vitro,and to map the reciprocal binding domains on each protein, the abilityof GST-ERα derivatives to bind to full-length DBC-1 or DBC-1 truncationfragments produced by in vitro translation was tested. DBC-1 bound mostefficiently to GST-ERα derivatives 1-595 (full-length ERα) and 302-595(Era hormone-binding domain). Reciprocally, GST-ERα 1-595 (full-lengthERα) bound to the extreme amino terminus of DBC-1 (amino acids 1-150).

To validate the ligand-independent interaction between DBC-1 and ERα invivo, a mammalian two-hybrid interaction analysis was employed. Chimericproteins consisting of DBC-1 fused to the GAL4-DNA-binding domain andERα fused to the VP16 activation domain were expressed with or withoutone another in HeLa cells and examined for their respective abilities toactivate transcription from a reporter template controlled by GAL4DNA-binding sites in both the absence and presence of E2. In the absenceof E2, DBC-1 and ERα exhibited a robust interaction that was disruptedby addition of E2 to the cell culture medium (FIG. 1A). Further analysisof DBC-1 amino and carboxyl truncation derivatives revealed that theligand-independent association between DBC-1 and ERα is mediatedentirely by the amino terminal half of DBC-1 (amino acids 1-150) (FIG.1B).

To confirm the ligand-independent in vivo association between DBC-1 andERα, the ability of antibodies specific for ERα or DBC-1 tocoprecipitate one another in MCF-7 human breast cancer cells, whichexpress both ERα and DBC-1, was examined. This analysis revealed thatDBC-1 was specifically and reciprocally co-immunoprecipitated along withunliganded, but not liganded, ERα, demonstrating that the two endogenousproteins interact in a strict ligand-independent manner in human breastcancer cells (FIG. 1C). The ligand independent interaction betweenendogenous DBC-1 and ERα was confirmed in both T-47D human breast andBG-1 human ovarian cancer cell lines, thus revealing the DBC-1/ERαinteraction to be conserved in a variety of ERα-expressing cell lines(FIG. 1D).

HSP90 together with additional heat shock family members andimmunophilins are known to form a heteromeric chaperone complex thatsequesters neosynthesized and unliganded ERα in an inactive state,primes it for ligand-binding, and protects it from proteolyticdegradation. The physical relationship between unliganded ERα in complexwith HSP90-based chaperones and DBC-1 was examined bycoimmunoprecipitation analysis using MCF-7 whole cell lysates. Whereasunliganded ERα immunoprecipitates included not only DBC-1, but alsoHSP90, DBC-1 immunoprecipitates included unliganded ERα, but neitherHSP90 nor the immunophilin CYP40 (FIG. 2A). Thus, DBC-1 is not acomponent of the classical HSP90-based molecular chaperone complex. Thesubcellular pool of unliganded ERα in specific association with DBC-1was identified by coimmunoprecipitation analysis using fractionatedMCF-7 cell lysates. ERα/DBC-1 complexes were found exclusively in thenuclear fraction (FIG. 2B), thus revealing that unliganded ERα isdistributed amongst at least two distinct protein complexes in humanbreast cancer cells, a cytosolic HSP90-based molecular chaperone complexand a nuclear DBC-1-containing protein complex.

The DBC-1 N-terminus Interacts Directly with the ERαHormone-BindingDomain in vitro. To determine if DBC-1 interacts directly withunliganded ERα, and to map the reciprocal binding domains on eachprotein, the ability of GST-ERα derivatives to bind to full-length DBC-1or DBC-1 truncation fragments produced by in vitro translation weretested. DBC-1 bound most efficiently to GST-ERα derivatives 1-595(full-length ERα) and 302-595 (ERα hormone-binding domain) (FIG. 3A).Reciprocally, GST-ERα 1-595 (full-length ERα) bound to the extremeamino-terminus of DBC-1 (amino acids 1-150) (FIG. 3B). Thus, in theabsence of ligand, the ERα hormone-binding domain can accommodate theDBC-1 amino terminus.

The DBC-1/ERα interface is a novel target of antiestrogens.Antiestrogens are currently the most widely administered endocrineagents for the management of ERα-expressing breast cancers.Antiestrogens competitively displace E2 from the ERα hormone-bindingdomain and either block ERα function or induce destabilization anddegradation of ERα. Tamoxifen, a prototype of the former class, is aselective estrogen receptor modulator (SERM) with antiestrogenicproperties in breast, and the most widely administered antiestrogen inbreast cancer therapy. Among the latter class of antiestrogens, ICI182,780 (FASLODEX® (fulvestrant), Astrazeneca, Wilmington, Del.) is aselective estrogen receptor downregulator (SERD) and an effectivetherapeutic agent used to treat breast cancers that have progressed onprior tamoxifen therapy. Because these compounds bind directly to theERα hormone-binding domain, the influence of each agent was examined onthe DBC-1/ERα interaction. The ability of ERα-specific antibodies toco-immunoprecipitate endogenous DBC-1 was tested in MCF-7 and BG-1 cellscultured in the absence or presence of E2, tamoxifen, or ICI 182,780.Like E2, both tamoxifen and ICI 182,780 disrupted the DBC-1/ERαinteraction (FIGS. 4A and 4B).

DBC-1 is an ERα-dependent prosurvival factor in breast cancer cells. Toexamine the biological consequence of the DBC-1/ERα interaction in humanbreast cancer cells, conditions for RNAi-mediated DBC-1 depletion wasestablished in MCF-7 cells. RNAi-mediated DBC-1 knockdown wasaccompanied by a significant reduction in the steady-state level of ERαprotein, but not ERα mRNA, suggesting that DBC-1 modulates ERα proteinsynthesis or stability (FIG. 5). Notably, DBC-1 knockdown preferentiallyreduced the steady-state level of unliganded, but not liganded, ERαprotein. Therefore, DBC-1 stabilizes unliganded ERα by virtue of theirdirect physical association (FIG. 5).

Because DBC-1 is a direct binding partner and key determinant ofsteady-state ERα protein levels, its role in ERα-dependent breast cancercell proliferation and survival was examined. RNAi-mediated DBC-1depletion significantly reduced E2-independent, but not E2-dependent,MCF-7 cell proliferation, an observation concordant with the fact thatDBC-1 preferentially binds to and modulates the levels of unliganded ERα(FIG. 6). Transient DBC-1 knockdown cells experienced an initial(˜2-fold) reduction in cell number on day 3 after siRNA deliveryfollowed by growth kinetics similar to control siRNA knockdown cells. Todetermine whether DBC-1 knockdown causes an increase in apoptotic celldeath, the influence of DBC-1 knockdown on the apoptotic fate of MCF-7cells cultured in the absence of E2 was examined. Under theseconditions, DBC-1 depletion increased the percentage of apoptotic cellsfrom 6.2% to 12.8%, thus revealing an anti-apoptotic function for DBC-1in the absence of hormone (FIG. 7A). To determine if DBC-1 promoteshormone-independent cell survival through its direct interaction withERα, the influence of DBC-1 knockdown on the apoptotic fate of MCF-7cells cultured in the presence of E2, which disrupts the DBC-1/ERαinteraction, or ICI 182,780, which not only disrupts the DBC-1/ERαinteraction but also drastically depletes ERα protein levels, wasexamined. Notably, DBC-1 depletion had no effect on MCF-7 cell apoptosisin the presence of either E2 or ICI 182,780 (FIG. 7A). Furthermore,DBC-1 depletion did not enhance apoptosis of ERα-negative MDAMB-231breast cancer cells cultured in the absence of E2 (FIG. 7B). Takentogether, these observations show that DBC-1 functions to promoteE2-independent breast cancer cell survival in an ERα-dependent manner.

Example 2 DBC-1 and ERα, are Expressed in Primary Human BreastCarcinomas

Consistent with the results above showing that DBC-1 modulates ERαexpression and survival activity, a strong association betweenexpression levels of DBC-1 and ERα was observed in primary human breastcarcinomas (FIG. 9 and Table 1). FIG. 9 shows pictures of representativeimmunohistochemical staining of DBC-1 in normal and cancerous breasttissues. FIG. 9 shows pictures of representative immunohistochemicalstaining of DBC-1 in normal and cancerous breast tissues. For Table 1,DBC-1, ERα, and PR protein levels were examined by immunohistochemistryin 88 breast tumor samples derived from the San Antonio Cancer Institutetissue bank (27 samples) and the Cooperative Human Tissue Network andthe Tissue Array Research Program (TARP) of the National CancerInstitute (TARP Breast and Ovarian Cancer Array) (61 samples). Tissuemicroarrays were deparafinized and rehydrated prior to immunostainingusing antibodies specific for ERα (ER 6F11, Novocastra/Vison BioSystems,Inc., Norwell, Mass.), PR(PR 636, DAKO, Carpinteria, Calif.), or DBC-1(mouse polyclonal antibody against DBC-1 amino acids 475-923). Stainedmicrroarrays were scanned into a Scanscope CS scanner system (AperioTechnologies, Vista, Calif.) and images were analyzed using the nuclearIHC algorithm bundled with the Aperio TMALab software (AperioTechnologies, Vista, Calif.). The relationships among stainingpercentages for each protein were compared using Spearman's rank ordercorrelation coefficient analysis. Statistical tests were two-sided.Table 1 shows that DBC-1, ERα, and progesterone receptor (PR) proteinlevels were all significantly positively correlated in primary humanbreast carcinomas.

TABLE 1 DBC-1, ERα, and progesterone receptor (PR) protein levels inprimary human breast carcinomas. ERα PgR DBC-1 PgR p < 0.001 1 (R =0.83)^(a) DBC-1 p < 0.001 p < 0.001 1 (R = 0.52) (R = 0.64) ^(a)Valuesin parentheses are Spearman rank correlation coefficients. Comparisonswherein p < 0.05 are statistically significant.

Together, these findings establish a principal biological function forDBC-1 in the modulation of ERα expression and hormone-independent breastcancer cell survival.

Example 3 DBC-1 is Required for Endocrine Resistant Breast Cancer CellSurvival and the DBC-1/ERα Complex is Resistant to Tamoxifen-MediatedDisruption in Tamoxifen-Resistant Cells

Since DBC-1 is a hormone-independent prosurvival factor in human breastcancer cells, its role in endocrine resistance was examined. Theexpression and function of DBC-1 was examined in a three-stage MCF-7cell-based model of acquired endocrine resistant breast cancer (Boukeret al., 2004, Cancer Res 64:4030-9; Brunner et al., 1997, Cancer Res57:3486-93; and Clarke et al., 1989, Proc Natl Acad Sci USA 86:3649-53).This model system is based on the ERα-positive MCF-7 human breast cancercell line, which is estrogen-dependent for growth and sensitive to thegrowth inhibitory actions of antiestrogens, including the selectiveestrogen receptor modulator (SERM) tamoxifen and the selective estrogenreceptor downregulator (SERD) ICI 182,780 (faslodex, fulvestrant).Long-term passage of MCF-7 tumor xenografts in ovariectomized mice ledto derivation of the MCF-7/LCC1 (LCC1) cell line, which isestrogen-independent but antiestrogen-sensitive (Clarke et al., 1989,Proc Natl Acad Sci USA 86:3649-53). Subsequent long-term culture of LCC1cells in vitro in the presence of ICI 182,780 produced the MCF7/LCC9(LCC9) cell line, which is fully resistant to both estrogen and ICI182,780, and cross-resistant to tamoxifen (Brunner et al., 1997, CancerRes 57:3486-93). This model system, derived through stepwise selectionof MCF-7 cells first to a low estrogen environment in vivo followed bylong-term culture in the presence of an antiestrogen, mimics a clinicalscenario [Phase II endocrine resistance (Jordan et al., 2005, Breast14:624-30)] in which breast cancer patients undergo exhaustive hormonaltherapy (first-line treatment with an aromatase inhibitor followed bysecond-line treatment with an antiestrogen) leading to the acquisitionof a fully estrogen-independent and antiestrogen-resistant tumorphenotype. Using this three-stage MCF-7 cell-based model of acquiredendocrine resistant breast cancer, it was determined that DBC-1 isupregulated during the acquisition of endocrine resistance and, further,that targeted suppression of DBC-1 triggers a rapid and profoundapoptotic response in endocrine resistant LCC1 and LCC9 breast cancercells (FIG. 10). It was also observed that a direct correlation existsbetween antiestrogen resistance and DBC-1/ERα complex formation in thismodel system. The DBC-1/ERα complex in tamoxifen-sensitive MCF-7 andLCC1 cells is disrupted by tamoxifen at a concentration (10⁻¹² M), fourorders of magnitude lower than that required to disrupt the DBC-1/ERαcomplex in tamoxifen-resistant LCC9 cells (10⁻⁸ M) (FIG. 11). These datashow that a direct correlation exists between tamoxifen resistant breastcancer cell growth and DBC-1/ERα complex formation. These data also showthat the DBC-1/ERα complex drives antiestrogen resistance through apro-survival pathway.

1. A method of determining whether a cancer cell is sensitive toendocrine therapy comprising: a) obtaining a population of cancer cells;and b) determining the level of expression or activity of Deleted inBreast Cancer-1 (DBC-1) in the cells, an increase in expression oractivity of DBC-1 as compared to a control indicating that the cancercells are not sensitive to endocrine therapy.
 2. The method of claim 1,wherein the cancer cells are endometrial cancer cells, breast cancercells, ovarian cancer cells or prostate cancer cells.
 3. The method ofclaim 1, wherein step b) is carried out by determining the level ofDBC-1 mRNA.
 4. The method of claim 1, wherein step b) is carried out bydetermining the level of DBC-1 protein.
 5. The method of claim 4,wherein step b) is carried out using a polyclonal antibody that bindsDBC-1 in the region of amino acids 475-923. 6.-8. (canceled)
 9. A methodof determining whether a subject with cancer is suitable for treatmentwith endocrine therapy comprising: a) obtaining a biological samplecomprising cancer cells from the subject; and b) determining the levelof expression or activity of DBC-1, an increase in expression oractivity of DBC-1 as compared to a control indicating that the subjectis not suitable for treatment with endocrine therapy.
 10. The method ofclaim 9, wherein the cancer cells are endometrial cancer cells, breastcancer cells, ovarian cancer cells or prostate cancer cells.
 11. Themethod of claim 9, wherein step b) is carried out by determining thelevel of DBC-1 mRNA.
 12. The method of claim 9, wherein step b) iscarried out by determining the level of DBC-1 protein.
 13. The method ofclaim 12, wherein step b) is carried out using a polyclonal antibodythat binds DBC-1 in the region of amino acids 475-923. 14.-16.(canceled)
 17. A method of determining a susceptibility to hormoneresistant cancer in a subject comprising: a) obtaining a biologicalsample comprising cancer cells from the subject; and b) determining thelevel of expression or activity of DBC-1, wherein an increase inexpression or activity of DBC-1 as compared to a control indicates thatthe subject is susceptible to hormone resistant cancer.
 18. The methodof claim 17, wherein the subject has endometrial cancer, breast cancer,ovarian cancer or prostate cancer.
 19. The method of claim 17, whereinstep b) is carried out by determining the level of DBC-1 mRNA.
 20. Themethod of claim 17, wherein step b) is carried out by determining thelevel of DBC-1 protein.
 21. The method of claim 20, wherein step b) iscarried out using a polyclonal antibody that binds DBC-1 in the regionof amino acids 475-923. 22.-24. (canceled)
 25. A method of inducingapoptosis of cancer cells comprising: a) selecting a population ofhormone resistant cancer cells, wherein the hormone resistant cancercells express ER-α; and b) contacting the hormone resistant cancer cellswith an agent that inhibits expression of DBC-1.
 26. The method of claim25, wherein the agent is an inhibitory nucleic acid.
 27. The method ofclaim 26, wherein the inhibitory nucleic acid is an siRNA. 28.-29.(canceled)
 30. The method of claim 25, wherein the cancer cells arebreast cancer cells, ovarian cancer cells or prostate cancer cells. 31.A method of treating hormone resistant cancer in a subject, comprising:a) selecting a subject with hormone resistant cancer, wherein the cellsof the hormone resistant cancer express ER-α; and b) administering tothe subject an agent that inhibits expression of DBC-1.
 32. The methodof claim 31, wherein the agent is administered in an effective amount toinduce apoptosis of the hormone resistant cancer cells.
 33. The methodof claim 31, wherein the subject has endometrial cancer, breast cancer,ovarian cancer or prostate cancer.
 34. The method of claim 31, whereinthe agent is comprised within a composition comprising the agent and apharmaceutically acceptable carrier.
 35. The method of claim 31, whereinthe agent is an inhibitory nucleic acid.
 36. The method of claim 35,wherein the inhibitory nucleic acid is an siRNA. 37.-38. (canceled) 39.The method of claim 31, further comprising administering achemotherapeutic agent to the subject.
 40. The method of claim 31,further comprising administering radiation to the subject.